The carbohydrate moieties of glycopeptide hormone human chorionic gonadotropin (hCG) have sialic acid (NeuAc) residues which are required for full biological activity. hCG stimulates adenylate cyclase in murine Leydig tumor MLTC-1 cells, and we used this system as a model to study the role of NeuAc in the biological activity of hCG. Removing NeuAc produced asialo-hCG which had only 50% of the biological activity of hCG. The NeuAct was replaced with a modified form of NeuAc or with the carbohydrate moiety of the ganglioside GM, which also contains NeuAc. Both derivatives had the same activity as the asialo-hCG indicating the importance of both position and structure of NeuAc for biological activity. The drug forskolin activates adenylate cyclase, but we found that different cells varied in their response to the drug as measured by cyclic AMP accumulation. Cyclic AMP accumulation in cells with a strong response to forskolin was blocked by activating the inhibitory G protein (Gi), with fluoroaluminate. Forskolin potentiated cyclic AMP accumulation stimulated by hormones that activate the stimulatory G protein (Gs), even in cells that responded poorly to forskolin alone. Activating Gi with fluoroaluminate prevented the hormone response, as well as its potentiation by forskolin. Forskolin stimulated adenylate cyclase activity in membranes, even if the membranes were prepared from cells that responded poorly to the drug. Activating Gi with GTP analogues inhibited forskolin-stimulated adenylate cyclase activity in membranes. The results indicate that interactions between Gs, Gi, and the catalytic subunit of adenylate cyclase affect the ability of forskolin to stimulate cyclic AMP accumulation. The observation that some cells responded well to forskolin and other responded poorly suggests that these interactions are not the same in all cell types. Furthermore, differences in the response of cells and their membranes to forskolin suggest that changes in these interactions occur during the preparation of membranes.